Microscopic system for mobile phase particle analysis and method thereof

Abstract

The invention relates to a microscopic system for mobile phase particle analysis and a method thereof. The microscope system is used for detecting a transparent sample and comprises a light source for generating light to illuminate a sample surface, and the sample surface is configured to carry the sample; comprising a pair of objective lenses with different numerical apertures, the objective lens group converges light from a light source to the sample surface and collects light from the sample surface, and the light from the sample surface comprises surrounding waves not diffracted with the transparent sample and diffracted waves diffracted with the transparent sample; the diaphragm is arranged to be adjacent to the objective lens group, the diaphragm is configured to adjust the proportion of the diffracted wave and the surrounding wave passing through the diaphragm, and the diffracted wave and the surrounding wave interfere on an imaging plane to generate a microscopic image of the transparent sample.

Description

Technical Field

[0001] The invention relates to a microscopic system and a method for analyzing mobile phase particles. Background Art

[0002] For non-luminous transparent objects, because there is a difference in refractive index between the non-luminous transparent object and the surrounding medium, light will be reflected and refracted at the interface between the two. It is through the difference between the reflected light or refracted light and the background light that the transparent object can be observed. However, in microscopic imaging, since the refractive index of the transparent object and the surrounding medium is relatively close, the transparent object is usually not easy to observe.

[0003] In the field of life science research, if you want to gain a deeper understanding of the life processes in cells (e.g., cells of yeast, algae, etc.), tissues, or organs, you need to first have a deep understanding of the structure and organization of the cells. However, most of these samples are colorless and transparent, and it is difficult to clearly observe their fine structures under a bright field illumination microscope. Therefore, stains and dyes are often used in the fields of biology and pharmacy to improve the visibility of biological tissues. Similarly, fluorescent probe labels have similar uses.

[0004] At present, macromolecular reagents such as trypan blue TB are used in bright field microscopy systems; small molecule reagents such as acridine orange AO / propidium iodide PI / 4',6-diamidino-2-phenylindole dihydrochloride DAPI are used in fluorescence microscopy systems. Both methods require pretreatment of cell samples, that is, using a specific ratio to fully mix the reagents, observe and compare under the microscopy system, and count the results. At present, a number of manual / automatic cell analyzers based on chemical staining or biomarker methods have been launched on the market worldwide.

[0005] However, whether it is staining, dyes or fluorescent probes, the samples need to be pretreated when performing microscopic observation. This process is complicated and will destroy the physiological state of the samples. Chemical staining or biomarkers are both biochemical methods. They always use exogenous methods to improve the imaging contrast of living cells and produce certain biochemical reactions on the cells themselves. They can neither monitor the cell state for a long time nor truly reflect the biophysical properties of the cells themselves. In addition, the introduction of reagents to pretreat the samples during the analysis process not only limits the detection speed and throughput, but also increases the complexity of the microscopic system. Summary of the invention

[0006] Therefore, one object of the present invention is to provide a microscopic system that can directly improve the endogenous contrast of cells without chemical or biological pretreatment of samples, thereby obtaining indicator results such as cell viability, diameter, and concentration; and can be combined with a liquid flow system for automatic and rapid sample loading to achieve fully automatic, high-throughput cell analysis functions.

[0007] According to some aspects of the present disclosure, a microscopic system for mobile phase particle analysis is provided, the microscopic system being configured to detect a transparent sample, the microscopic system comprising a light source, the light source being configured to generate light to illuminate a sample surface, the sample surface being configured to carry a sample; an objective lens group, the objective lens group comprising a pair of objective lenses with different numerical apertures, the sample surface being located between the two objective lenses, the objective lens group being configured to converge light from the light source to the sample surface and to collect light from the sample surface, wherein the light from the sample surface comprises an ambient wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample; and an aperture, the aperture being disposed adjacent to the objective lens group, the aperture being configured to adjust a ratio of the diffracted wave and the ambient wave passing therethrough, wherein the diffracted wave and the ambient wave interfere at an imaging surface to generate a microscopic image of the transparent sample.

[0008] In some embodiments, the aperture includes an annular light-passing portion and a central light-shielding portion, wherein the central light-shielding portion corresponds to a surround wave that is not diffracted by the transparent sample, and the annular light-passing portion corresponds to a diffracted wave that is diffracted by the transparent sample, wherein the central light-shielding portion allows the surround wave to pass with attenuation, and the annular light-passing portion allows the diffracted wave to pass with essentially no attenuation.

[0009] In some embodiments, the contrast C of the microscopic image of the transparent sample is related to the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0010] In some embodiments, the contrast of the microscopic image of the transparent sample is in is the luminous flux of the diffracted wave passing through the aperture, is the luminous flux of the surround wave passing through the aperture.

[0011] In some embodiments, the contrast C of the microscopic image of the sample satisfies a predetermined condition by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0012] In some embodiments, the contrast ratio 1≤C≤10 is adjusted by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0013] In some embodiments, the objective lens group includes a first objective lens and a second objective lens along an optical axis from the sample plane to the imaging plane, the first objective lens being configured to converge light from a light source to the sample plane, and the second objective lens being configured to collect light from the sample plane.

[0014] In some embodiments, the first objective lens has a first numerical aperture and a first focal length, the second objective lens has a second numerical aperture and a second focal length different from the first numerical aperture, when the first numerical aperture is smaller than the second numerical aperture, the aperture is arranged along the optical path behind the second objective lens, wherein when the first numerical aperture is larger than the second numerical aperture, the aperture is arranged along the optical path before the first objective lens.

[0015] In some embodiments, the distance between the first objective lens and the sample plane is equal to a first focal length, and the distance between the second objective lens and the sample plane is equal to a second focal length.

[0016] In some embodiments, the objective lens with a larger numerical aperture in the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA b and focal length f b , wherein the objective lens with a smaller numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA s and focal length f s , where the cross-sectional area of ​​the central light shielding portion of the aperture corresponding to the surround wave is S block =π·(NA s *f b ) 2 The cross-sectional area of ​​the annular light-passing portion of the aperture corresponding to the diffraction wave is S ring =π·(NA b *f b ) 2 -π·(NA s *f b ) 2 .

[0017] In some embodiments, the microscope system further includes a collimating lens group disposed between the light source and the objective lens group, wherein the collimating lens group is configured to collimate the light from the light source into a parallel light beam to feed the objective lens group.

[0018] In some embodiments, the microscope system further includes an imaging lens disposed between the objective lens group and the imaging plane, and an imaging component disposed at the imaging plane, wherein the objective lens group is configured to converge light collected from the sample plane into parallel light, and the imaging lens is configured to converge the parallel light to the imaging component at the imaging plane.

[0019] In some embodiments, the imaging mirror includes a first positive lens and a first negative lens that are adjacently arranged.

[0020] In some embodiments, the central light shield of the aperture allows less than or equal to 30% of the surround waves to pass through the aperture.

[0021] In some embodiments, the transparent sample does not need to be chemically stained or biolabeled prior to being viewed.

[0022] In some embodiments, the microscope system further comprises a control unit connected to the aperture, wherein the control unit is configured to adjust a ratio of the diffracted wave and the surrounding wave passing through the aperture.

[0023] In some embodiments, the sample surface includes a fluid channel for carrying the sample, and the fluid channel is connected in series with a fluid source, a pump, and a recovery vessel to achieve automatic sample loading.

[0024] According to other aspects of the present disclosure, a method for microscopic imaging for mobile phase particle analysis is also disclosed, the method being used to detect transparent samples, the method comprising: a light source generating light to illuminate a sample surface, the sample surface being configured to carry a sample; an objective lens group converging light from the light source onto the sample surface and collecting light from the sample surface, wherein the light from the sample surface comprises an ambient wave transmitted from the transparent sample and a diffraction wave scattered by the transparent sample, the objective lens group comprising a pair of objective lenses with different numerical apertures, the sample surface being located between the two objective lenses, and an aperture adjacent to the objective lens group adjusting a ratio of the diffraction wave and the ambient wave passing through the aperture, wherein the diffraction wave and the ambient wave interfere on an imaging surface to generate a microscopic image of the transparent sample.

[0025] According to other aspects of the present disclosure, a readable storage medium is also disclosed, including computer program instructions: when the computer program instructions are executed by at least one processor of an electronic device, the microscopic imaging method as described above is implemented.

[0026] According to other aspects of the present disclosure, an electronic device is also disclosed, including a memory and a processor; the memory is configured to store computer program instructions; the processor is configured to execute the computer program instructions to implement the microscopic imaging method as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other purposes and advantages of the present disclosure are further described below in conjunction with specific embodiments and with reference to the accompanying drawings. In the accompanying drawings, the same or corresponding technical features or components will be represented by the same or corresponding reference numerals.

[0028] Figure 1 A schematic diagram of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention;

[0029] Figure 2 Schematic diagram of a microscopic system for mobile phase particle analysis according to some other embodiments of the present invention;

[0030] Figure 3A schematic diagram of an aperture of a microscope system for mobile phase particle analysis according to some embodiments of the present invention;

[0031] Figure 4 is a schematic diagram for calculating the luminous flux passing through an aperture according to some embodiments of the present invention;

[0032] Figure 5A and Figure 5B A schematic diagram of an application scenario of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention;

[0033] Fig. 6A and 6B Microscopic images of a microscopic system for mobile phase particle analysis under different contrasts C according to some embodiments of the present invention; Figure 6C Microscopic images of conventional microscope systems;

[0034] Figure 7 is a schematic diagram of an electronic device according to some embodiments of the present invention. DETAILED DESCRIPTION

[0035] The following detailed description is made with reference to the accompanying drawings, and the following detailed description is provided to assist in a comprehensive understanding of the various exemplary embodiments of the present disclosure. The following description includes various details to assist in understanding, but these details are considered to be examples only and not to limit the present disclosure, which is defined by the appended claims and their equivalents. The words and phrases used in the following description are only used to enable a clear and consistent understanding of the present disclosure. In addition, for the sake of clarity and brevity, descriptions of well-known structures, functions, and configurations may be omitted. Those of ordinary skill in the art will recognize that various changes and modifications may be made to the examples described herein without departing from the scope of the present disclosure.

[0036] Biological research often requires the observation of thin and transparent biological samples, such as living cells, microorganisms, thin tissue sections, etc. However, because the samples are thin and transparent, it is difficult to see them directly under an optical microscope. Therefore, when observing cells, a specific dye is often used to stain the samples to improve the visibility of biological tissues. However, both chemical staining and biological markers belong to biochemical methods, and they always use exogenous methods to improve the imaging contrast of living cells and produce certain biochemical reactions on the cells themselves. They can neither monitor the cell state for a long time nor truly reflect the biophysical properties of the cells themselves. In addition, the sample pretreatment that requires the introduction of reagent reactions during the analysis process limits the detection speed and throughput; it also increases the complexity of the microscopic system.

[0037] Biological research therefore requires a method for directly observing cells without pretreatment, and the microscopic system for mobile phase particle analysis according to some embodiments of the present invention solves this problem. Under the microscopic system according to the present invention, living cells can be examined in a natural state without being killed, fixed, and stained in advance. Therefore, the dynamics of ongoing biological processes can be observed and recorded.

[0038] The microscope system of the present invention adopts a single light source illumination method, and by adding a diaphragm with a specific structure to the microscope system, the incident light is split into two components: the first component is a surround wave (S wave) with basically no deviation (no diffraction; zero order), which passes through the sample and surrounds the sample but does not interact with the sample; the second component is a diffraction wave (D wave) with deviation (diffraction), which passes through the sample and interacts with the internal structure of the sample and diffracts in all directions. After the surround wave and the diffracted wave enter the objective lens, they are projected to different spatial positions of the rear focal plane of the objective lens, and the S wave and the D wave are amplitude modulated by the diaphragm with a specific structure at the rear focal plane; then the S wave and the D wave are converged to the image plane through the imaging lens and synthesized into a particle wave P wave, and the synthesized particle wave P wave is completely generated by the interference of the separated surround wave front and the diffraction wave front.

[0039] By controlling the ratio of the component intensities (square of the amplitude) of different surround waves and diffracted waves on the aperture sheet, that is, the light flux of the S wave and the D wave, different phase enhancement or attenuation effects are obtained, thereby enhancing the refractive index differences of different organelles in the cell, as well as the intracellular and extracellular organelles. The microscope system according to the present invention can directly improve the endogenous contrast of the cell without chemical or biological pretreatment of the sample, and can reflect the slight refractive index differences inside and outside the cell or between different cells, such as the slight refractive index differences between dead cells and living cells, thereby obtaining the results of indicators such as cell viability, diameter, and concentration; and can be combined with the liquid flow system for automatic and rapid sample loading to achieve fully automatic, high-throughput cell analysis functions.

[0040] Figure 1 Schematic diagram of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention. Figure 1 As shown, the microscope system 100 includes a light source 101, a collimating lens group 102, an objective lens group 103, a sample surface 104, an aperture 105, an imaging lens 106, and an imaging surface 107. The sample to be observed is placed at the sample surface 104. Exemplary samples include but are not limited to cells, microorganisms, or thin tissue sections. In one embodiment of the present invention, the sample is transparent and does not need to be chemically stained or biologically labeled before being observed.

[0041] In the following description, the direction from the light source 101 to the sample plane 104 and then to the imaging plane 107 is considered to be the optical axis of the microscope system 100, that is, the Z direction. The direction perpendicular to the Z direction is considered to be the X direction. The direction perpendicular to both the Z direction and the X direction is considered to be the Y direction. The sample stage (not shown) of the sample plane 104 is in the plane formed by the X direction and the Y direction.

[0042] The light source 101 is configured to generate light to illuminate a sample surface 104. The sample is placed at a sample stage of the sample surface 104. The light source 101 can have any configuration, such as a halogen lamp or an LED lamp. The collimating lens group 102 is configured to collimate the light from the light source 101 into a parallel light beam. The collimating lens group 102 can have any configuration, such as a combination of a plano-convex lens and a pair of convex lenses arranged oppositely. The light source 101 is arranged at the focus of the collimating lens group 102. The objective lens group 103 is arranged after the collimating lens group 102 along the optical axis Z.

[0043] The objective lens group 103 is configured to converge the collimated light from the light source 101 onto the sample plane and collect the light from the sample plane, wherein the light from the sample plane 104 includes an undeflected surround wave (S wave), which passes through the sample and surrounds the sample but does not interact with the sample; and also includes a deflected diffraction wave (D wave), which passes through the sample and interacts with the internal structure of the sample and diffracts in various directions.

[0044] The objective lens group 103 is provided with a first objective lens 1031 and a second objective lens 1032 in sequence along the optical axis. The distance between the first objective lens and the second objective lens is the sum of the respective focal lengths of the first objective lens and the second objective lens. The sample plane 104 is arranged between the first objective lens 1031 and the second objective lens 1032. The distance between the sample plane 104 and the first objective lens 1031 is the focal length of the first objective lens 1031, and the focal length between the sample plane 104 and the second objective lens 1032 is the focal length of the second objective lens 1032. The first objective lens 1031 is configured to converge the light from the light source 101 to the sample plane 104, and the second objective lens 1032 is configured to collect the light from the sample plane 104. The light collected from the sample plane 104 by the second objective lens 1032 as described in the previous paragraph is divided into two parts. The surround wave (S wave) indicated by the letter S and the corresponding auxiliary dotted line is a light beam formed by the light focused by the first objective lens 1031 onto the sample surface passing through the sample. The surround wave passes through the sample and surrounds the sample but does not interact with the sample. The second part indicated by the letter D and the corresponding auxiliary dotted line is the diffraction wave (D wave) around the surround wave. The diffraction wave passes through the sample and interacts with the internal structure of the sample, diffracting in all directions. Since the diffraction wave interacts with the internal structure of the sample, the diffraction wave will be delayed in phase compared to the surround wave. In addition, because the sample surface 104 is located at the focal plane of the second objective lens 1032, both the diffraction wave and the surround wave will be collimated by the second objective lens 1032 into parallel light beams and fed to the subsequent aperture 105.

[0045] The aperture 105 is arranged adjacent to the objective lens group 103 along the optical axis Z, and the aperture is configured to adjust the ratio of the surrounding wave and the diffracted wave passing therethrough. When the numerical aperture of the first objective lens 1031 is smaller than the numerical aperture of the second objective lens 3032, that is, Figure 1 In the arrangement shown, the aperture 105 is arranged behind the second objective lens 1032 along the optical axis. On the contrary, since the light source, the sample plane and the imaging plane constitute conjugate planes in pairs, and based on the principle of reversibility of the optical path, as shown in FIG. Figure 2 As shown in FIG. 2 , when the numerical aperture of the first objective lens 2031 is greater than the numerical aperture of the second objective lens 2032, the diaphragm 205 will be arranged before the first objective lens 2031 along the optical axis. Figure 2 That is, the aperture 105 is always arranged to be close to the objective lens with a larger numerical aperture in the objective lens group 103, because the rear focal plane of the objective lens with a larger numerical aperture has relatively complete surround wave and diffraction wave information. Figure 1In the embodiment of the present invention, the aperture 105 is arranged behind the second objective lens 1032 along the optical axis. Generally, the light intensity of the central surround wave emitted from the second objective lens 1032 is higher than the light intensity of the surrounding diffracted wave emitted from the second objective lens 1032. Figure 3 As will be explained more clearly, the aperture 105 is configured to block a large portion of the central surround waves, allowing only a small portion of the central surround waves to pass through the aperture 105, while allowing the surrounding diffracted waves to almost all pass through the aperture 105. Figure 1 , the aperture 105 blocks a large portion of the central surround wave represented by S, while allowing almost all of the surrounding diffraction waves represented by D to pass. Figure 1 and Figure 2 The aperture 105 and the aperture 205 shown in FIG. 1 are only schematic. The specific geometric structures of the aperture 105 and the aperture 205 are shown in FIG. 1 . Figure 3 As shown in . Therefore, the aperture 105 at the rear focal plane of the second objective lens 1032 is configured to modulate the ratio of the surround wave and the diffraction wave from the objective lens group passing therethrough through its special geometric structure because it receives relatively complete surround wave and diffraction wave information at the same time, thereby adjusting the ratio between the high-frequency signal (i.e., diffraction wave) representing the detail features of the observed sample and the low-frequency signal (i.e., surround wave) representing the contour features of the observed sample. By mixing the high-frequency signal and the low-frequency signal in a certain ratio, a high-definition presentation of a transparent sample is achieved, thereby realizing the observation of living cells in a natural state without the need to fix and stain the living cells in advance. By adjusting the passing ratio of the diffraction wave and the surround wave, the final imaging effect can also be adjusted, such as choosing whether to highlight the internal details or the external contour.

[0046] Figure 2 Schematic diagram of a microscopic system for mobile phase particle analysis according to some other embodiments of the present invention. Figure 2 As shown, when the numerical aperture of the first objective lens 2031 is greater than the numerical aperture of the second objective lens 2032, the aperture 205 will be arranged before the first objective lens 2031 along the optical axis. In short, the aperture is always arranged to be close to the objective lens with a larger numerical aperture in the objective lens group. The aperture 205 is configured to block a large part of the central surround wave represented by S, allowing only a small part of the surround wave to pass through the aperture 205, while allowing all the surrounding light (represented by the letter D) to be diffracted by the sample to pass through and enter the second objective lens 2032 as a diffracted wave. Figure 1 Similarly, the aperture 105 Figure 2 The aperture 205 is arranged to modulate the ratio of the diffracted wave and the surrounding wave by its special geometric structure.

[0047] Figure 3Schematic diagram of the aperture 305 of the microscopic system for mobile phase particle analysis according to some embodiments of the present invention. For the convenience of description and illustration, the aperture 305 is drawn together with the objective lens group 303 including the first objective lens 3031 and the second objective lens 3032 and the sample surface 304. In order to clearly show the relationship between different parts of the aperture 305 and the numerical aperture and focal length of the two objective lenses, Figure 3 The viewing direction of the aperture in is the Z direction, while the viewing directions of the first objective lens and the second objective lens are the X direction. Figure 3 In the embodiment of FIG. 3 , the aperture 305 is disposed adjacent to the objective lens group 303. Specifically, in Figure 3 In the embodiment, the first objective lens 3031 along the optical path has a smaller numerical aperture, and the second objective lens 3032 has a larger numerical aperture. Based on the above description, the aperture 305 is arranged to be adjacent to the objective lens 3032 with a larger numerical aperture in the objective lens group. It is clear that the technical solution of the present invention also includes the case where the first objective lens 3031 has a larger numerical aperture and the second objective lens 3032 has a smaller numerical aperture. According to the reversible principle of the optical path, based on Figure 2 The same geometrical settings of the aperture are also applicable to Figure 3 Description of the aperture structure and explanation of the principle.

[0048] like Figure 3 As shown, the aperture 305 includes a central light shielding portion 3051 and an annular light-passing portion 3052. In some embodiments of the present invention, the transmittance of the central light shielding portion 3051 is adjustable to adjust the ratio of the diffracted wave and the surrounding wave. In some embodiments of the present invention, the central light shielding portion 3051 has a translucent film, and the translucent film allows a small part of the surrounding light incident on the central light shielding portion to pass through, for example, less than or equal to 30% of the surrounding light. In some embodiments of the present invention, the central light shielding portion 3051 is formed of an electrochromic material, and the transmittance of the central light shielding portion 3051 can be adjusted by an external electric field. In some embodiments of the present invention, the central light shielding portion 3051 includes a plurality of through holes dispersed therein or a single through hole located in the center to allow only a small part of the surrounding light to pass through. In some embodiments of the present invention, the through holes include but are not limited to square, circular, rectangular, triangular, trapezoidal, etc.

[0049] The parallel light is received from the second objective lens 3032 located before the aperture 305, and the beam of parallel light includes the central surround wave S and the surrounding diffracted wave D. The aperture 305 is configured to block a large part of the central surround wave S, allowing only a small part of the surround wave S to pass through, while allowing the surrounding diffracted wave D to pass through substantially all. The area of ​​the central light shielding portion 3051 corresponds to the circular projection area of ​​the surround wave S on the second objective lens 3032, and the area of ​​the annular light-passing portion 3052 corresponds to the annular projection area of ​​the diffracted wave D on the second objective lens 402. It can be seen that the area of ​​the central light shielding portion 3051 corresponds to the light transmitted from the sample without offset, and the area of ​​the annular light-passing portion 3052 corresponds to the light diffracted from the sample with offset. In some embodiments of the present invention, the central light shielding portion 3051 of the aperture can also be connected to the outer portion of the aperture 305 through one or more connecting portions (not shown). The one or more connecting portions span the annular light-passing portion 3052.

[0050] According to the principle of trigonometric function, the area of ​​the central light shielding portion 3051 of the aperture 305 is:

[0051] S S ≤π·(NA s *f b ) 2 Formula 1

[0052] where NA s For an objective lens with a small numerical aperture (i.e. Figure 3 The numerical aperture of the objective lens 3031) in b For an objective lens with a large numerical aperture (i.e. Figure 3 The focal length of the objective lens 3032).

[0053] The area of ​​the annular light-passing portion 3052 of the aperture 305 is

[0054] S D =π·(NA b *f b ) 2 -π·(NA s *f b ) 2 Formula 2

[0055] where NA b For an objective lens with a large numerical aperture (i.e. Figure 3 The numerical aperture of the objective lens 3032) in s For an objective lens with a small numerical aperture (i.e. Figure 3 The numerical aperture of the objective lens 3031) in b For an objective lens with a large numerical aperture (i.e. Figure 3 The focal length of the objective lens 3032).

[0056] It should be noted that since the aperture is arranged adjacent to the objective lens with a larger numerical aperture, both the area of ​​the central light shielding portion 3051 of the aperture and the area of ​​the annular light passing portion 3052 of the aperture are equal to the focal length f of the objective lens 3032 with a larger numerical aperture. b The focal length f of the objective lens with a small numerical aperture is s Not relevant.

[0057] In some embodiments of the present invention, when the numerical aperture of the first objective lens along the optical path is greater than the numerical aperture of the second objective lens, the aperture 305 will be arranged before the first objective lens along the optical path, and Formulas 1 and 2 are still applicable to the calculation of the area of ​​the central light-shielding portion and the annular light-passing portion of the aperture 305.

[0058] Reference again Figure 1 , after passing through the aperture 105, the diffracted wave and the surround wave are converged to the imaging surface 107 by the imaging lens 106. The diffracted wave is diffracted by the sample (for example, a cell), and a certain phase delay is imposed on the substance in the sample. In contrast, although the surround wave also passes through the inside of the sample, it basically does not interact with the substance in the sample, so the surround wave basically has no phase delay. As a result, the diffracted wave and the surround wave constitute coherent light with a phase difference from each other, so that the diffracted wave and the surround wave interfere at the imaging surface 107 to generate a microscopic image of the sample with a higher light-dark contrast, thereby directly improving the endogenous contrast of the cell without chemical or biological pretreatment of the sample, thereby obtaining the results of indicators such as cell viability, diameter, and concentration.

[0059] Furthermore, since the surround wave usually has a relatively strong brightness, if the surround wave and the diffraction wave are directly interfered, the stronger surround wave signal will drown out the weaker diffraction wave signal, making it impossible to reflect the relatively weak changes in the amplitude and direction of the diffraction wave. Therefore, the present invention reduces the light flux of the surround wave by using an aperture with a central light shielding portion, and uses a combination of a specially configured objective lens group and an aperture to increase the ratio of the light flux of the diffraction wave to the light flux of the surround wave, thereby increasing the sensitivity of the microscope system to relatively weak refractive index changes in transparent samples. By blocking most of the surround waves and allowing almost all of the diffraction waves to pass, the diffraction wave Φ can be adjusted. D and surround wave Ф S The ratio of the light flux to the contrast ratio C needs to satisfy the following formula 3, so that the different organelles in the cell and the relatively weak refractive index differences inside and outside the cell can be observed, making the edge of the transparent sample clearer and improving the observation effect:

[0060]

[0061] Among them Ф Dis the luminous flux of the diffracted wave passing through the aperture; Ф S is the luminous flux of the surrounding wave passing through the aperture. As shown in Formula 3, preferably, the contrast C is between 1 and 10.

[0062] Figure 4 FIG. 4 is a schematic diagram for calculating the luminous flux Φ involving the aperture 400 according to some embodiments of the present invention.

[0063] Referring to Formula 4, the unit luminous flux dΦ is equal to the unit solid angle dω multiplied by the luminous intensity per unit area L·ds·cosU. Integrating Formula 4 over the solid angle of the aperture gives Formula 5, where L is the illumination intensity of the light source, and sinU is the numerical aperture of the objective lens with a larger numerical aperture next to the aperture.

[0064] dФ=L·ds·cosU·dω=L·ds·cosU·sinU dU·dθ Formula 4

[0065] Ф=L·ds·∫∫cosU·sinU dU·dθ=π·L·ds·sin 2 U Formula 5

[0066] Substituting the area of ​​the central light shielding part of the aperture into Formula 5 and multiplying it by the light flux attenuation coefficient will give Ф S , that is, the luminous flux of the center surround wave passing through the aperture; Substituting the area of ​​the annular light-passing part of the aperture into formula 5 will give Ф D , that is, the luminous flux of the surrounding diffracted waves passing through the aperture. s and the luminous flux of the diffracted wave Φ D Substituting into Formula 3, we obtain the contrast expression C of the final image. Preferably, the contrast C is not greater than 10 and not less than 1. Preferably, the contrast C is 1.

[0067] Back to Figure 1, the microscope system 100 further includes an imaging lens 106 disposed between the objective lens group 103 and the imaging plane 107. The second objective lens 1032 in the objective lens group 103 is configured to converge the light collected from the sample surface 104 into parallel light, which includes the surround wave at the center and the diffracted wave located around the surround wave. The imaging lens 106 is configured to converge the parallel light to the imaging component at the imaging plane 107. In other words, the surround wave and the diffracted wave, which are coherent light, will be converged to the imaging component at the focal plane of the imaging lens. In one embodiment of the present invention, the imaging lens 106 includes a first positive lens and a second negative lens disposed adjacent to each other. A curved surface of the first positive lens is in contact with a curved surface of the second negative lens. The microscope system 100 further includes an imaging plane 107 disposed at the exit light path of the imaging lens 106, and the imaging plane 107 includes an imaging component, wherein the diffracted wave and the surround wave are interfered and imaged at the imaging component. The imaging component 107 may include a charge coupled device (CCD) or a complementary metal oxide semiconductor device (CMOS). In some embodiments, the optical detection device may also include a memory, a processor, etc. that are communicatively connected to the imaging component to automatically store and process the image information formed by the imaging component, thereby further facilitating detection.

[0068] Figure 5A and Figure 5B They are schematic diagrams of application scenarios of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention. Figure 5A and Figure 5B In some embodiments of the present invention, the sample is placed in a channel located at the sample surface 504. In some embodiments of the present invention, the channel is a fluid channel. The channel is combined with a fluid flow system including a fluid source 509, a pump 511 and a recovery vessel 510 to automatically and quickly load the sample, thereby realizing a fully automatic, high-throughput cell analysis function, so that the microscopic system can observe the microscopic image of the transparent sample flowing through the fluid channel in real time. Figure 5A In the embodiment of the present invention, the numerical aperture of the first objective lens 5031 is larger than the numerical aperture of the second objective lens 5032, and the aperture 505 is therefore located before the first objective lens 5031 having the larger numerical aperture along the optical path. Figure 5B In the embodiment of the present invention, the numerical aperture of the first objective lens 5031 is smaller than the numerical aperture of the second objective lens 5032, and the aperture 505 is therefore located after the second objective lens 5032 having a larger numerical aperture along the optical path. Figure 5A and Figure 5BIn the embodiment of the present invention, the structure of the aperture 505 is designed to allow almost all diffracted waves to pass through the aperture, while only a small part of the surround waves to pass through the aperture. The diffracted waves and the surround waves are collected by the second objective lens 5032 and converged to the imaging component at the imaging surface 507 through the imaging lens 506 to form a microscopic image of the sample. The processor 508 is respectively connected to the optical devices before and after the sample surface 504 to obtain imaging information at the imaging surface 507 and / or control the aperture 505 and / or control the light source 501. Optionally, the ratio of the diffracted waves and the surround waves passing through the aperture 505 is automatically or semi-automatically adjusted according to the imaging information, for example, by adjusting the transmittance of the electrochromic material of the central light shielding portion of the aperture 505, or for example, the size, number and / or shape of the through holes of the central light shielding portion (not shown) of the aperture 505. Optionally, the position of the aperture 505 in three-dimensional space is automatically or semi-automatically adjusted according to the imaging information to substantially align the annular light-transmitting portion and the central light-shielding portion with the diffracted waves and the surround waves, respectively.

[0069] Fig. 6A and 6B ] are microscopic images of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention at different contrasts C. In contrast, Figure 6C This is a microscopic image from a conventional microscope system. Fig. 6A , Figure 6B and Figure 6C The imaging is performed for the same object field of view. According to Formula 3, when the surround wave is attenuated to a certain extent, for example, when the contrast C=1 is reached, the following is obtained: Fig. 6A The microscopic image shown in FIG. 1 shows a transparent sample with a significantly brighter outline relative to the background. When the surround wave is further attenuated, for example, when the contrast ratio C=10 is reached, the image shown in FIG. 1 is obtained. Figure 6B Microscopic image shown, where the transparent sample appears as a significantly darker outline relative to the background.

[0070] Figure 7 Schematic diagram of the structure of an electronic device provided according to some embodiments of the present disclosure. Figure 7As shown, the electronic device 700 provided in this embodiment includes: a memory 701 and a processor 702. Among them, the memory 701 can be an independent physical unit, and the processor 702 can be connected through a bus 703. The processor 702 may include but is not limited to a CPU, a hardware microprocessor, a hardware processor, a multi-core processor, a single-core processor, a microcontroller, an application-specific integrated circuit (ASIC), a DSP or other similar processing devices, and can execute any type of instructions, algorithms or software for the operation and function of microscopic imaging according to the embodiments described in this disclosure. The processor 702 can be various implementations of a digital circuit system, an analog circuit system or a mixed signal (a combination of analog and digital) circuit system that performs functions in a computing system. The processor 702 may include, for example, an integrated circuit (IC), a portion or circuit of a separate processor core, an entire processor core, a separate processor, a programmable hardware device such as a field programmable gate array (FPGA), and / or a system including multiple processors. The processor 702 may be a central processing unit (CPU), a network processor (NP) or a combination of a CPU and a NP. The processor 702 may further include a hardware chip. The hardware chip may be an application-specific integrated circuit (ASIC), a programmable logic device (PLD) or a combination thereof. The PLD may be a complex programmable logic device (CPLD), a field-programmable gate array (FPGA), a generic array logic (GAL) or any combination thereof.

[0071] The memory 701 can be integrated with the processor 702 and implemented by hardware, etc. The memory 701 is used to store program instructions, and the processor 702 calls the program instructions to perform the operations of any of the above method embodiments. The memory 701 may include a volatile memory (volatile memory), such as a random-access memory (random-access memory, RAM); the memory may also include a non-volatile memory (non-volatile memory), such as a flash memory (flash memory), a hard disk drive (hard disk drive, HDD) or a solid-state drive (solid-state drive, SSD); the memory may also include a combination of the above types of memory. The memory 701 can be used to store any type of instructions, software or algorithms, including instructions for controlling the general functions and operations of electronic devices.

[0072] Optionally, when part or all of the methods of the above embodiments are implemented by software, the above electronic device 700 may also only include a processor 702. The memory 701 for storing programs is located outside the electronic device 700, and the processor 702 is connected to the memory through circuits / wires to read and execute the programs stored in the memory.

[0073] The present disclosure may be implemented as any combination of an apparatus, a system, an integrated circuit, and a computer program or a program product on a non-transitory computer-readable medium.

[0074] It should be understood that the computer executable instructions in the computer readable storage medium or program product according to the embodiments of the present disclosure can be configured to perform operations corresponding to the above-mentioned device and method embodiments. When referring to the above-mentioned device and method embodiments, the embodiments of the computer readable storage medium or program product are clear to those skilled in the art, so they are not repeatedly described. Computer readable storage media and program products for carrying or including the above-mentioned computer executable instructions also fall within the scope of the present disclosure. Such storage media may include, but are not limited to, floppy disks, optical disks, magneto-optical disks, memory cards, memory sticks, and the like.

[0075] In addition, it should be understood that the above series of processes and devices can also be implemented by software and / or firmware. In the case of being implemented by software and / or firmware, the storage medium of the relevant device stores the corresponding program constituting the corresponding software, and when the program is executed, various functions can be performed.

[0076] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0077] The words "front", "rear", "top", "bottom", "above", "below", etc., if present, in the specification and claims are used for descriptive purposes and are not necessarily used to describe invariant relative positions. It should be understood that the words so used are interchangeable under appropriate circumstances, such that the embodiments of the disclosure described herein, for example, are capable of operation in other orientations than those illustrated or otherwise described herein.

[0078] As used herein, the word "exemplary" means "serving as an example, instance, or illustration," rather than as a "model" to be precisely copied. Any implementation described herein as exemplary is not necessarily to be construed as superior to other implementations.

[0079] Furthermore, the present disclosure is not limited by any expressed or implied theory given in the above technical field, background technology, summary of the invention or detailed description.

[0080] As used herein, the term "substantially" is intended to include any minor variations due to design or manufacturing imperfections, device or component tolerances, environmental influences, and / or other factors. The term "substantially" also allows for deviations from a perfect or ideal condition due to parasitic effects, noise, and other practical considerations that may exist in actual implementations.

[0081] The description may indicate an element or node or feature that is "connected" or "coupled" together. As used herein, unless otherwise expressly stated, "connected" means that one element / node / feature is directly connected (or directly communicates) with another element / node / feature electrically, mechanically, logically or otherwise. Similarly, unless otherwise expressly stated, "coupled" means that one element / node / feature can be mechanically, electrically, logically or otherwise connected to another element / node / feature in a direct or indirect manner to allow interaction, even if the two features may not be directly connected. In other words, "coupled" is intended to include direct and indirect connections of elements or other features, including connections using one or more intermediate elements.

[0082] It should also be understood that when the term “include / comprises” is used in this document, it indicates the presence of the specified features, integers, steps, operations, units and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, units and / or components and / or their combinations.

[0083] Those skilled in the art will appreciate that the boundaries between the above operations are merely illustrative. Multiple operations can be combined into a single operation, a single operation can be distributed in additional operations, and operations can be performed at least partially overlapping in time. Moreover, alternative embodiments may include multiple instances of specific operations, and the order of operations may be changed in other various embodiments. However, other modifications, variations, and replacements are equally possible. Therefore, this specification and accompanying drawings should be considered illustrative, not restrictive.

[0084] Although some specific embodiments of the present disclosure have been described in detail by way of example, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present disclosure. The various embodiments disclosed herein may be combined in any manner without departing from the spirit and scope of the present disclosure. It should also be understood by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

1. A microscopic system for mobile phase particle analysis, the microscopic system being configured to detect a transparent sample, the microscopic system comprising: a light source configured to generate light to illuminate a sample surface configured to carry a sample; an objective lens group, the objective lens group comprising two objective lenses with different numerical apertures, the sample plane being located between the two objective lenses, the objective lens group being configured to converge light from a light source to the sample plane and to collect light from the sample plane, wherein the light from the sample plane comprises a surround wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, and an aperture, the aperture being disposed adjacent to the objective lens group, the aperture being configured to adjust a ratio of the diffracted wave and the surrounding wave passing therethrough, The diffracted wave and the surrounding wave passing through the aperture interfere with each other on an imaging surface to generate a microscopic image of the transparent sample.

2. The microscope system according to claim 1, wherein the aperture comprises an annular light-passing portion and a central light-shielding portion, the central light-shielding portion corresponds to a surround wave that is not diffracted by the transparent sample, and the annular light-passing portion corresponds to a diffracted wave that is diffracted by the transparent sample, wherein the central light-shielding portion allows the surround wave to pass with attenuation, and the annular light-passing portion allows the diffracted wave to pass with essentially no attenuation.

3. The microscope system according to claim 1, wherein the contrast C of the microscope image of the transparent sample at the imaging plane is related to the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

4. The microscopic system according to claim 3, wherein the contrast of the microscopic image of the transparent sample at the imaging surface is in is the luminous flux of the diffracted wave passing through the aperture, is the luminous flux of the surround wave passing through the aperture. 5 . The microscope system according to claim 4 , wherein the contrast C of the microscope image of the sample satisfies a predetermined condition by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

6. The microscope system according to claim 5, wherein the contrast ratio 1≤C≤10 is adjusted by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

7. The microscope system according to claim 1, wherein the objective lens group includes a first objective lens and a second objective lens along an optical axis from the sample plane to the imaging plane, the first objective lens being configured to converge light from a light source to the sample plane, and the second objective lens being configured to collect light from the sample plane.

8. A microscope system according to claim 7, wherein the first objective lens has a first numerical aperture and a first focal length, the second objective lens has a second numerical aperture and a second focal length different from the first numerical aperture, when the first numerical aperture is smaller than the second numerical aperture, the aperture is arranged along the optical path after the second objective lens, wherein when the first numerical aperture is larger than the second numerical aperture, the aperture is arranged along the optical path before the first objective lens.

9. The microscope system according to claim 7, wherein the distance between the first objective lens and the sample surface is equal to a first focal length, and the distance between the second objective lens and the sample surface is equal to a second focal length.

10. The microscope system according to claim 7, wherein the objective lens with a larger numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA b and focal length f b , wherein the objective lens with a smaller numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA s and focal length f s , where the cross-sectional area of ​​the central light shielding portion of the aperture corresponding to the surround wave is S S ≤π·(NA s *f b ) 2 The cross-sectional area of ​​the annular light-passing portion of the aperture corresponding to the diffraction wave is S D =π•(NA b *f b ) 2 -π•(NA s *f b ) 2 .

11. The microscope system according to claim 1, wherein the microscope system further comprises a collimating lens group disposed between the light source and the objective lens group, the collimating lens group being configured to collimate the light from the light source into a parallel light beam to be fed to the objective lens group.

12. The microscope system according to claim 1, wherein the microscope system further comprises an imaging lens arranged between the objective lens group and the imaging plane, and an imaging component arranged at the imaging plane, wherein the objective lens group is configured to converge the light collected from the sample plane into parallel light, and the imaging lens is configured to converge the parallel light to the imaging component at the imaging plane.

13. The microscope system according to claim 11, wherein the imaging lens comprises a first positive lens and a first negative lens which are adjacently arranged.

14. The microscope system of claim 1, wherein a central light shielding portion of the aperture allows less than or equal to 30% of the surround waves to pass through the aperture.

15. The microscopy system of claim 1, wherein the transparent sample does not need to be chemically stained or biologically labeled before being observed. 16 . The microscope system according to claim 1 , further comprising a control unit connected to the aperture, the control unit being configured to adjust a ratio of the diffracted wave and the surrounding wave passing through the aperture.

17. The microscopy system according to claim 1, wherein the sample surface comprises a fluid channel for carrying the sample, and the fluid channel is connected in series with a fluid source, a pump, and a recovery vessel to achieve automatic sample loading.

18. A method for microscopic imaging of mobile phase particle analysis, the method being applied to detect transparent samples, the method comprising: a light source generating light to illuminate a sample face, the sample face being configured to carry a sample; an objective lens group that converges light from a light source onto the sample surface and collects light from the sample surface, wherein the light from the sample surface includes a surrounding wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, the objective lens group includes a pair of objective lenses with different numerical apertures, and the sample surface is located between the two objective lenses; and The aperture next to the objective lens group adjusts the ratio of the diffracted wave and the surrounding wave passing through the aperture. The diffracted wave and the surrounding wave interfere at an imaging plane to generate a microscopic image of the transparent sample.

19. A readable storage medium comprising computer program instructions: When the computer program instructions are executed by at least one processor of an electronic device, the microscopic imaging method of claim 18 is implemented.

20. An electronic device comprising a memory and a processor; The memory is configured to store computer program instructions; The processor is configured to execute the computer program instructions to implement the microscopic imaging method of claim 18.

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